We have analyzed the expression of Snail1 protein in 162 tumours obtained from colon cancer patients (). This analysis was carried out using a specific MAb that only detects one band in western blot, reacts with Snail1 protein and not with Snail2 
. The specificity of this antibody for the analysis of paraffin-embedded sections was demonstrated by the morphological location of the positive cells detected in embryonic samples and also by the lack of immunoreactivity in sections from Snail1 KO embryos 
. Expression of Snail1 was detected in 128 of the 162 tumours analysed (79%) and not in the normal tissue obtained from distal areas of the same patients (). A tumour was considered positive when at least 1% of the cells in the analyzed area showed Snail1 staining. This threshold was chosen in order to compare ours results with previous analysis in other tumours using this cut-off 
. Only cells with nuclear reactivity were considered to be positive. Cytosolic staining was occasionally detected in our analyses in epithelial cells. This cytosolic reactivity was not considered since, although it may be due to a residual expression of Snail1 protein, this transcriptional factor has been shown to be inactive in the cytosol 
, rendering its expression outside the nucleus irrelevant.
Characteristics of 162 patients with colorectal cancer.
Expression of Snail1 in tumor and stroma according to tumor stage.
Representative sections obtained in our analysis are shown in . The number of positive cells in the different samples was variable; from tumours with few Snail1 expressing cells () to some cases were Snail1 was massively expressed (). Nuclear immunoreactivity was normally associated with areas of invasion, but not all the invasion fronts were positive. Nuclear expression of Snail1 was more abundant in the stroma. Reactivity in this compartment was observed in spindle fibroblast-like cells and also in histiocytes detected in areas of inflammation. In 34 cases () the immunoreactivity was observed only in the stroma and tumour cells were negative for Snail1 expression. In most cases, positive stromal cells were placed close to the tumoral cells (). In 90 samples, positive cells were observed both in the tumour and in the stroma (). Frequently, nuclear expression of Snail1 in tumour cells corresponded to areas where the tumour was losing its epithelial structure (). In some cases, it was not possible to determine if the immunoreactive cell was a carcinoma or stromal cell (for instance, see labelled cell at ).
Nuclear Snail1 protein expression in colon carcinomas.
Some other examples of cells presenting Snail1 nuclear expression are also shown. For instance, we detected Snail1 immunoreactive cells migrating out of degenerating glands () or in the glandular lumen (). In some occasions Snail1 expressing cells seemed to be entering a vessel, as the labelled cell in . Reactivity in endothelial cells were often detected, as shown in . The high expression of Snail1 detected in areas of inflammation () was also remarkable. Although not common (only 4 cases), the presence of nuclear reactivity in the tumour but not in the stroma was also observed (see ). These four samples showed a very low number of positive cells.
We analyzed if Snail1 nuclear expression correlated with clinicopathologic features. shows the presence of Snail1 in the tumour and stroma of colorectal tumours at different stages. No significant correlation was found between the expression of Snail1 in the tumour and the tumour stage when we compared columns T+/S− and T+/S+ versus T−/S− and T−/S+ in . However, when we considered immunoreactivity in the stroma (T−/S+ and T+/S+ versus T+/S− and T−/S−), a trend was obtained with regard to the tumour stage, with a p
0.053. Since all stage IV tumours presented Snail1-positive cells in the stroma (), a correlation (p
0,006) was established between the presence of distant metastasis at the moment of the diagnosis and Snail1 immunoreactivity in the stroma. No significant associations were observed between Snail1 expression in any of the two compartments and other parameters (lymph node metastasis, degree of differentiation or tumour site).
The correlation between Snail1 expression and patients' survival was also determined. Specific survival was determined since this parameter reflects the nature of cancer more accurately than overall survival.). As expected according to the higher expression observed in stage IV tumours, presence of Snail1 in the stroma correlated with a lower survival (p
0.011, see , left panel). Additional immunoreactivity in the tumour did not decrease the p value (not shown), giving further indication that the presence of Snail1 in the stroma was the most relevant parameter. Moreover, lower survival was also observed when we compared negative tumours with tumours showing Snail1 reactivity only in the stroma (, right panel).
Kaplan-Meier specific survival curves for colon carcinoma patients according to Snail1 expression in the stroma.
We also determined whether Snail1 expression in the stroma of tumours of different stages also correlated with lower survival, regardless its presence in the tumour. This analysis could not be carried out in stage IV tumours since all our specimens were positive for Snail1. We did not find a significant correlation in stage III tumours; however the association between Snail1 immunoreactivity in the stroma and lower survival was significant for stage I and II tumours (). Unfortunately, more elaborated statistical analysis could not be performed due to the lack of events in Snail1 negative tumours. However, a Kaplan-Meier analysis was also performed after classifying Snail1-positive samples according to the degree of expression, determined as indicated in Methods
. This analysis was only performed on stage II tumours and also demonstrated that presence of Snail1 protein in the stroma correlated with lower survival ().
Specific survival of stage I, II and III colon tumour patients according to Snail1 expression in the stroma.